Recently, the cell cultures with the purpose of the development of artificial organs and the development of regeneration medicine, safety evaluation, drugs and the like are noted and their techniques are variegated and have become more and more highly advanced. As an example, the co-culture method (coexistent culture) for culturing two or more kinds of cells on the same culture substrate can be mentioned.
Heretofore, cell culture has been conducted on a glass surface or on the surface of a synthetic polymer having been subjected to various treatments. For example, various vessels using polystyrene as the material which have been subjected to the treatment, for example, γ-ray irradiation and silicone coating are widespread as the cell culture vessels but none of them could sufficiently cope with various culture methods.
Japanese Patent Publication (Kokoku) No. Hei 6-104061/1994 describes a novel cell culture method comprising culturing cells on a cell culture substrate whose substrate surface has been coated with a polymer having an upper or lower critical solution temperature to water of 0 to 80° C. at a temperature of not higher than the upper critical solution temperature or not lower than the lower critical solution temperature, and then rendering the culture temperature a temperature of not lower than the upper critical solution temperature or not higher than the lower critical solution temperature to detach the cultured cells without treating with an enzyme. However, with the substrate illustrated herein, a temperature-responsive polymer is uniformly coated on the substrate surface, and accordingly all cultured cells on the substrate surface are detached, and this substrate could not fully cope with a wide variety of culture methods.
Further, it is very difficult to allow a plurality of kinds of cells to proliferate to effect co-culture by using the above described cell culture vessels. For example, co-culture (in consideration of the structure of the liver of the body of living) has been considered to be effective for the long-term culture of hepatocyte but it is impossible to effect the co-culture by simply mixing hepatocyte with fibroblasts or hepatocyte with endothelial cells.
Hepatocyte is by no means singly present in the liver. The liver is composed of a repetition of a fine structure of a so-called hepatic lobule which endothelial cells, stellate cells and the like in addition to the hepatocyte constitutes. Co-culture has been examined on the basis of the thought that hepatic nonparenchymal cells are important for the maintenance of the function of hepatocyte. Further, also from the observation that the hepatic differentiation function can be better maintained when hepatic nonparenchymal cells are included, the necessity of hepatic nonparenchymal cells are strongly suggested when the primary hepatocyte is incorporated into an artificial liver module. However, the method using conventional dishes realizes only an ultrashort-term co-culture alone and, in addition, except for the variation in the ratio of the number of both kinds of cells, a sufficient examination could not be done and the details could not be clarified.
Toner et al. fixed laminin in the form of a pattern on a silicone surface by irradiation with rays with the use of the photoresist technique [S. N. Bhatia, M. L. Yarmush and M. Toner, J. Biomed. Mater. Res., 34, 189-199 (1997)]. When hepatocyte was cultured on this surface, they attached only to the laminin-fixed domain to proliferate. Then, when fibroblasts were seeded, they attached on to the silicone domain due to their very high adhesion to achieve a co-culture system with the hepatocyte. However, any combination could not achieve the co-culture system, and the co-culture of hepatocyte with endothelial cells having not so high adhesion as that of fibroblast could not be achieved.
Japanese Patent Publication (Kokai) No. Hei 4-94679/1992 developed the technology as illustrated in the above described Japanese Patent Publication (Kokoku) No. Hei 6-104061/1994 and described a method for culturing cells with the use of a substrate whose surface has been coated with a temperature-responsive polymer in the form of a pattern. However, the substrate as illustrated herein had weak cell affinity for its surface to which the temperature-responsive polymer had not attached, and was insufficient as the substrate for culturing a plurality of kinds of cells. As long as this substrate was used, it was impossible to detach an entire cell sheet extending over the substrate surface after the co-culture.